Long term preservation is crucial as an enabling technology for regenerative medicine products that contain living cells. However, while cryopreservation works well for most cells, some cells are more sensitive and hard to cryopreserve by freezing. Plus, the three dimensional structure and extracellular architecture of tissues is damaged by ice formation that results from more conventional cryopreservation protocols that use freezing. Vitreous cryopreservation is an alternative cryopreservation strategy that stabilizes the tissue as a glass, no ice crystallization. However, there is the risk of cytotoxicity if the sample is not managed correctly during both addition and removal of the high concentrations of cryoprotectants required for vitrification. Avoidance of high concentration cryoprotectant formulations by reduction of cryoprotectant concentrations while reducing or inhibiting ice formation may be possible by mimicking the strategy employed by certain insects to survive sub-zero temperatures. These insects combine increases in cryoprotectant content (glycerol) with the onset of cold environmental conditions with production of several antifreeze peptides (AFPs) to survive temperatures as low as -80[unreadable]C. This feasibility study is to determine the potential benefits of using these insect AFPs to reduce or inhibit ice formation for the cryopreservation of cells that do not cryopreserve well under conventional conditions. The ability of these AFPs to control ice both inside and outside the cell will be investigated. Establishment of a protocol that produces >75% viability after cryopreservation will then be optimized and applied to tissues in Phase II. PUBLIC HEALTH RELEVANCE: Long term storage technologies are necessary for newly developed cell-based therapies. Cryopreservation is the most obvious avenue for storage but ice formation in the cells and tissues can cause irreparable damage. The use of high concentrations of cryoprotectants would facilitate avoidance of ice but at the price of cytotoxicity. This proposal will develop preservation protocols using antifreeze proteins such that less cryoprotectants are required and ice formation is minimized or eliminated. This strategy would provide preservation methods for cell and tissue transplants that may eventually impact more than a 100 million US patients.